Probes for detection of UGT1A1 gene, reagent containing the same, and the uses thereof

ABSTRACT

Primer sets for amplifying target regions containing sites to be detected in the UGT1A1 gene by a gene amplification method are provided, wherein the primer sets can amplify the regions specifically Three pairs of primer sets are used including forward primers consisting of the base sequences of SEQ ID NOs: 4 or 81, 21, and 42 as well as reverse primers consisting of the base sequences of SEQ ID NOs: 13 or 91, 29 and 48, respectively. The use of these primer sets makes it possible to amplify three target regions including parts where three types of polymorphisms (UGT1A1*6, UGT1A1*27, and UGT1A1*28) of the UGT1A1 gene are generated, respectively, in the same reaction solution at the same time.

RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 12/293,954, filed Sep. 22, 2008 now U.S. Pat. No. 8,357,516, whichis a U.S. National Phase Application of International Application No.PCT/JP2007/073107, filed Nov. 29, 2007, which claims the benefit ofJapanese Patent Application No. 2006-322955, filed Nov. 30, 2006 andJapanese Patent Application No. 2007-232611, filed Sep. 7, 2007.

TECHNICAL FIELD

The present invention relates to primer sets for amplifying the UGT1A1gene, reagents for amplifying the UGT1A1 gene containing the same, andthe uses thereof.

BACKGROUND ART

UDP-Glucuronosyl Transferase (UGT) is an enzyme that catalyzes thereaction of adding glucuronic acid to, for example, a drug, a foreignsubstance, or an endogenous substance such as bilirubin, steroidhormone, or bile acid. It is known as a drug-metabolizing enzyme. Aplurality of isozymes that are classified into the UGT1 family and theUGT2 family have been reported as the UGT. Genetic polymorphisms existin a gene (UGT1A1 gene) that codes UGT1A1 belonging to the UGT1 familyamong those isozymes. Generally, they are said to be involved in theincidence of side effects of irinotecan hydrochloride, an anticanceragent. Specifically, it has been reported that when a patient has aUGT1A1 gene polymorphism, the function of detoxifying irinotecanhydrolysate (SN-38) having high antitumor activity by using the UGT isdeteriorated, which causes serious side effects such as a decrease inthe number of white blood cells and diarrhea. Known examples of typicalgenetic polymorphisms involved in such side effects include UGT1A1*28, apolymorphism in a promoter region, as well as UGT1A1*6 and UGT1A1*27,polymorphisms in exon 1. Particularly, it has been reported that inaddition to UGT1A1*28, the most important polymorphism, Asians includingJapanese also have at least one of polymorphisms, UGT1A1*6 andUGT1A1*27, in combination therewith and thereby stronger side effectstend to be manifested. Furthermore, since UGT1A1 is involved inbilirubin conjugation formed through glucuronic acid in vivo, thepolymorphisms thereof also cause constitutional jaundice such as Gilbertsyndrome. Accordingly, the examination of a plurality of polymorphismswith respect to the UGT1A1 gene is very important to predict the degreeand the onset of side effect to be caused by an anticancer agent.

On the other hand, the detection of a point mutation, a so-called singlenucleotide polymorphism (SNP), is employed widely as a method ofanalyzing, at the gene level, for example, the causes of all types ofdiseases and the individual differences in disease liability(susceptibility to diseases) and in drug action. Examples of the commonmethods of detecting a point mutation include: (1) a direct sequencingmethod in which the region corresponding to a sequence to be detected ina target DNA of a sample is amplified by a polymerase chain reaction(PCR) and all the gene sequences are analyzed, (2) a RFLP analysis inwhich the region corresponding to a sequence to be detected in a targetDNA of a sample is amplified by PCR, the amplification product thusobtained is cut with a restriction enzyme whose cleaving action differsdepending on the presence or absence of the target mutation in thesequence to be detected and is then electrophoresed, and thereby typingis performed, and (3) the ASP-PCR method in which PCR is performed usinga primer with a target mutation located at the 3′-end region and themutation is judged depending on the presence or absence ofamplification.

However, since these methods require, for example, purification of DNAextracted from a sample, electrophoresis, and a treatment with arestriction enzyme, they take time and cost. Furthermore, after PCR isperformed, it is necessary to open the reaction container once.Accordingly, there is a possibility that the amplification product maycontaminate the next reaction system and thereby the analysis accuracymay be deteriorated. Moreover, since it is difficult to automate, alarge amount of samples cannot be analyzed. Further, the aforementionedASP-PCR method (3) is less specific, which also is a problem.

Because of these problems, recently, a method of analyzing the meltingtemperature (Tm) of double-stranded nucleic acid formed of a probe andtarget nucleic acid is used as a method of detecting a point mutation.Since such a method is performed through, for example, Tm analysis oranalysis of the melting curve of the double strand, it is referred to asmelting curve analysis. This method is described below. That is, first,a probe complementary to a sequence to be detected containing a targetpoint mutation is used to form a hybrid (double-stranded DNA) betweenthe aforementioned probe and a target single-stranded DNA contained in adetection sample. Subsequently, this hybridization product isheat-treated, and dissociation (melting) of the hybrid accompanying thetemperature rise is detected by a change in a signal such as absorbance.The Tm value then is determined based on the result of the detection andthe presence or absence of any point mutation is judged accordingly. Thehigher the homology of the hybridization product, the higher the Tmvalue, and the lower the homology, the lower the Tm value. Therefore theTm value (reference value for assessment) is determined beforehand withrespect to the hybridization product between the sequence to be detectedcontaining a point mutation and a probe complementary thereto, and thenthe Tm value (measured value) of the hybridization product between thetarget single-stranded DNA contained in the detection sample and theaforementioned probe is measured. When the measured value is comparableto the reference value, it is considered as matching, that is, it can bejudged that a point mutation is present in the target DNA. On the otherhand, when the measured value is lower than the reference value, it isconsidered as mismatching, that is, it can be judged that no pointmutation is present in the target DNA. Furthermore, according to thismethod, it also is possible to automate gene analysis.

However, such a detection method using Tm analysis also has a problem inthat a region including a site to be detected must be able to beamplified specifically and efficiently in PCR. Particularly, manyisozymes are present in UGT and the sequences for coding them also arevery similar to one another. Accordingly, there is a possibility thatgenes coding isozymes other than UGT1A1 also are amplified in PCR.Furthermore, when other isozyme-coding genes also have been amplified asdescribed above, it may cause a decrease in the reliability of theanalysis result in the analysis of a particular polymorphism (UGT1A1*28,UGT1A1*6, or UGT1A1*27) of the UGT1A1 gene (Nonpatent Document 1).Moreover, as described above, since the analysis of one sample isaccompanied by a considerable amount of time and energy, it is notpractical to analyze a large amount of samples, which also is a problem.

-   [Nonpatent Document 1] PMID: 11156391 Cancer Res. 2000 Dec. 15;    60(24): 6921-6.

DISCLOSURE OF INVENTION

Hence, the present invention is intended to provide primer sets forspecifically amplifying a target region in the UGT1A1 gene by a geneamplification method.

In order to achieve the aforementioned object, a primer set of thepresent invention is a primer set for amplifying the UGT1A1 gene by agene amplification method, wherein the primer set includes at least oneselected from the group consisting of the following primer sets (1) to(3):

Primer set (1):

a primer set of a pair of primers including a forward primer composed ofthe following oligonucleotide (F1) and a reverse primer composed of thefollowing oligonucleotide (R1):

-   (F1): at least one oligonucleotide selected from:

oligonucleotide that is at least one oligonucleotide having a sequenceidentical to that of a region extending from adenine (A) at base 2120 tobe considered as the first base to any one of the 18^(th) to 22^(nd)bases in the direction toward the 5′ end in the UGT1A1 gene consistingof the base sequence of SEQ ID NO: 1, with the adenine (A) being the 3′end, and

oligonucleotide that is at least one oligonucleotide having a sequenceidentical to that of a region extending from cytosine (C) at base 2140to be considered as the first base to any one of the 18^(th) to 34^(th)bases in the direction toward the 5′ end in the UGT1A1 gene consistingof the base sequence of SEQ ID NO: 1, with the cytosine (C) being the 3′end,

-   (R1): at least one oligonucleotide selected from:

oligonucleotide that is at least one oligonucleotide complementary to aregion extending from guanine (G) at base 2226 to be considered as thefirst base to any one of the 17^(th) to 27^(th) bases in the directiontoward the 3′ end in the UGT1A1 gene consisting of the base sequence ofSEQ ID NO: 1, with cytosine (C) complementary to the guanine (G) at base2226 being the 3′ end, and

oligonucleotide that is at least one oligonucleotide complementary to aregion extending from cytosine (C) at base 2198 to be considered as thefirst base to any one of the 22^(nd) to 39^(th) bases in the directiontoward the 3′ end in the UGT1A1 gene consisting of the base sequence ofSEQ ID NO: 1, with guanine (G) complementary to the cytosine (C) at base2198 being the 3′ end,

Primer set (2):

a primer set of a pair of primers including a forward primer composed ofthe following oligonucleotide (F2) and a reverse primer composed of thefollowing oligonucleotide (R2):

-   (F2): oligonucleotide that is at least one oligonucleotide having a    sequence identical to that of a region extending from guanine (G) at    base 2622 to be considered as the first base to any one of the    15^(th) to 27^(th) bases in the direction toward the 5′ end in the    UGT1A1 gene consisting of the base sequence of SEQ ID NO: 1, with    the guanine (G) being the 3′ end, and-   (R2): oligonucleotide that is at least one oligonucleotide    complementary to a region extending from cytosine (C) at base 2687    to be considered as the first base to any one of the 17^(th) to    26^(th) bases in the direction toward the 3′ end in the UGT1A1 gene    consisting of the base sequence of SEQ ID NO: 1, with guanine (G)    complementary to the cytosine (C) at base 2687 being the 3′ end, and    Primer set (3):

a primer set of a pair of primers including a forward primer composed ofthe following oligonucleotide (F3) and a reverse primer composed of thefollowing oligonucleotide (R3):

-   (F3): oligonucleotide that is at least one oligonucleotide having a    sequence identical to that of a region extending from cytosine (C)    at base 1863 to be considered as the first base to any one of the    17^(th) to 31^(st) bases in the direction toward the 5′ end in the    UGT1A1 gene consisting of the base sequence of SEQ ID NO: 1, with    the cytosine (C) being the 3′ end, and-   (R3): oligonucleotide that is at least one oligonucleotide    complementary to a region extending from cytosine (C) at base 1928    to be considered as the first base to any one of the 16^(th) to    20^(th) bases in the direction toward the 3′ end in the UGT1A1 gene    consisting of the base sequence of SEQ ID NO: 1, with guanine (G)    complementary to the cytosine (C) at base 1928 being the 3′ end.

A reagent for amplifying a gene of the present invention is a reagentfor amplifying the UGT1A1 gene by a gene amplification method, whereinthe reagent includes the primer set for amplifying the UGT1A1 gene ofthe present invention.

A method of manufacturing an amplification product of the presentinvention is a method of manufacturing an amplification product of theUGT1A1 gene by a gene amplification method, wherein the method includesthe following step (I):

(I) amplifying the UGT1A1 gene in a reaction solution using a primer setfor amplifying the UGT1A1 gene according to the present invention, withnucleic acid contained in a sample being used as a template.

A polymorphism analysis method of the present invention is a method ofanalyzing a polymorphism of a site to be detected in the UGT1A1 gene,wherein the method includes the following steps (i) to (iv):

(i) amplifying a region including a site to be detected in the UGT1A1gene in a reaction solution by a method of manufacturing anamplification product of the present invention,

(ii) preparing a reaction solution that contains the amplificationproduct obtained in step (i) and a probe capable of hybridizing to thesite to be detected,

(iii) measuring signal values that indicate melting states of ahybridization product between the amplification product and the probewhile changing the temperature of the reaction solution, and

(iv) determining a polymorphism of the site to be detected from a changein the signal values accompanying a change in the temperature.

The primer set of the present invention makes it possible specificallyand efficiently to amplify a target region in a reaction solution, withthe target region including the site where a polymorphism to be detected(UGT1A1*28, UGT1A1*6, or UGT1A1*27) is generated in the UGT1A1 gene.Accordingly, the time and cost can be reduced, which is different fromthe conventional methods described above. Furthermore, as describedabove, since a region including a site to be detected where a specificpolymorphism of the UGT1A1 gene is generated can be amplifiedspecifically, for example, further the use of a probe complementary to asequence to be detected including the site to be detected makes itpossible to perform Tm analysis by directly using the aforementionedreaction solution to type the polymorphism. Moreover, sinceamplification of the target region and typing of the polymorphism can beperformed with one reaction solution, it is also possible to automatethe operation. Since the use of the primer set of the present inventionallows a pretreatment to be omitted even in the case of, for example, acontaminated sample (for instance, whole blood or oral mucosa), theamplification reaction can be carried out quicker and more simply.Furthermore, since the use of the primer set of the present inventionallows the amplification reaction to be carried out with higheramplification efficiency as compared to the conventional case, theamplification reaction time also can be shortened. Thus, according tothe primer set of the present invention and a reagent including the sameas well as the method of manufacturing an amplification product and apolymorphism analysis method, in each of which the primer set and thereagent are used, polymorphisms in the UGT1A1 gene can be analyzedquickly and simply, and it therefore can be said that they are veryeffective in the field of medicine.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows graphs indicating the results of Tm analysis in Example 1of the present invention.

FIG. 2 shows graphs indicating the results of Tm analysis in Example 1of the present invention described above.

FIG. 3 shows graphs indicating the results of Tm analysis in Example 1of the present invention described above.

FIG. 4 shows graphs indicating the results of Tm analysis in Example 2of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

<Primer Set for Amplifying UGT1A1 Gene>

As described above, the primer set for amplifying the UGT1A1 gene of thepresent invention is characterized by including at least one primer setselected from the group consisting of the aforementioned primer sets (1)to (3). The inclusion of at least one of the primer sets makes itpossible, for example, to amplify specifically a specific target regionin the UGT1A1 gene.

The primer set for amplifying the UGT1A1 gene of the present inventionmay include, for example, one of the aforementioned primer sets (1) to(3) or may include two or all of the primer sets (1) to (3). Asdescribed later, the target region that can be amplified specificallywith the primer set (1) is a region including a site where thepolymorphism UGT1A1*6 is generated in the UGT1A1 gene; the target regionthat can be amplified specifically with the primer set (2) is a regionincluding a site where the polymorphism UGT1A1*27 is generated in theUGT1A1 gene; and the target region that can be amplified specificallywith the primer set (3) is a region including a site where thepolymorphism UGT1A1 *28 is generated in the UGT1A1 gene.

As described above, since these three types of polymorphisms in theUGT1A1 gene are known as polymorphisms that affect drug metabolism, itis considered to be important to examine not only one of them but two orall of the three types of polymorphisms. However, the conventionalmethods have a problem in that a plurality of sequences cannot beanalyzed in one reaction system. Conceivably, as described above, thisis because the many isozymes exist in a UGT and thereby genes codingisozymes other than UGT1A1 also are amplified in PCR. Accordingly, inorder to examine two or all of the three types of polymorphisms(UGT1A1*28, UGT1A1*6, and UGT1A1*27) in the UGT1A1 gene, it is necessarythat the regions including the sites where the respective polymorphismsare generated are amplified in separate reaction systems, respectively,and the resultant amplification products are analyzed separately. Thus,with the conventional methods, it is very difficult to use only theUGT1A1 gene selected from the UGT genes as a template and to amplifyspecifically only two or three types of target regions including thesites where polymorphisms are generated, respectively, in the UGT1A1gene. Furthermore, since such analysis of even one sample is accompaniedby a considerable amount of work, there is a problem in that theanalysis of a large amount of samples is not practical. On the contrary,according to the primer set for amplifying the UGT1A1 gene of thepresent invention, even in the case where two or all of the three typesof the primer sets (1) to (3) are included, the respective targetregions can be amplified in the same reaction solution simultaneouslyand specifically. Accordingly, the time and cost can be reduced, whichis different from the aforementioned conventional methods. Furthermore,since two or three target regions are amplified specifically in the samereaction solution as described above, for example, the use of a probecomplementary to a sequence to be detected in each target region makesit possible to perform Tm analysis directly using the aforementionedreaction solution to type each of the two or three types ofpolymorphisms. As described above, since two or three types ofpolymorphisms in the UGT1A1 gene can be analyzed in the same reactionsolution, it is suitable for the primer set for amplifying the UGT1A1gene of the present invention not only to include one of the primer sets(1) to (3) but also to include two or three of them. When not only onetarget region but also two or three target regions are amplifiedsimultaneously using such a UGT1A1 gene primer set, polymorphisms in theUGT1A1 gene can be analyzed more efficiently as compared to theconventional cases.

Hereinafter, a forward primer also may be referred to as “F primer” anda reverse primer as “R primer”.

As described above, the primer set (1) is a primer set of a pair ofprimers including a forward primer composed of the followingoligonucleotide (F1) and a reverse primer composed of the followingoligonucleotide (R1):

-   (F1): at least one oligonucleotide selected from:

oligonucleotide that is at least one oligonucleotide having a sequenceidentical to that of a region extending from adenine (A) at base 2120 tobe considered as the first base to any one of the 18^(th to) 22^(nd)bases in the direction toward the 5′ end in the UGT1A1 gene consistingof the base sequence of SEQ ID NO: 1, with the adenine (A) being the 3′end, and

oligonucleotide that is at least one oligonucleotide having a sequenceidentical to that of a region extending from cytosine (C) at base 2140to be considered as the first base to any one of the 18^(th) to 34^(th)bases in the direction toward the 5′ end in the UGT1A1 gene consistingof the base sequence of SEQ ID NO: 1, with the cytosine (C) being the 3′end,

-   (R1): at least one oligonucleotide selected from:

oligonucleotide that is at least one oligonucleotide complementary to aregion extending from guanine (G) at base 2226 to be considered as thefirst base to any one of the 17^(th) to 27^(th) bases in the directiontoward the 3′ end in the UGT1A1 gene consisting of the base sequence ofSEQ ID NO: 1, with cytosine (C) complementary to the guanine (G) at base2226 being the 3′ end, and

oligonucleotide that is at least one oligonucleotide complementary to aregion extending from cytosine (C) at base 2198 to be considered as thefirst base to any one of the 22^(nd) to 39^(th) bases in the directiontoward the 3′ end in the UGT1A1 gene consisting of the base sequence ofSEQ ID NO: 1, with guanine (G) complementary to the cytosine (C) at base2198 being the 3′ end.

The base sequence indicated in SEQ ID NO: 1 is a full-length sequence ofthe UGT1A1 gene of human (Homo sapiens)-derived UGT1 family and, forexample, has been registered at NCBI under the accession No. AY603772.

The primer set (1) is a primer set for amplifying a DNA strand includingany one of a region from base 2121 to base 2225, a region from base 2141to base 2197, a region from base 2121 to 2197, and a region from base2141 to 2225 in SEQ ID NO: 1, as well as a strand complementary thereto.Base 2160 in this region (i.e. base 2160 in SEQ ID NO: 1) is known forthe presence of a point mutation (2160G, 2160A) that affects thefunction of UGT1A1, and the polymorphism thereof is UGT1A1*6 describedabove. In the present invention, the polymorphism of this site can beindicated as 2060G/G or 2160A/A in the case of homozygote and as 2160G/Ain the case of heterozygote. Hereinafter, this primer set (1) also maybe referred to as a “primer set for UGT1A1*6”. When only thepolymorphism UGT1A1*6 is to be analyzed, it is sufficient to use onlythe primer set for UGT1A1*6.

In the present invention, the F1 primer and R1 primer of the primer set(1) can be any primers, as long as the base located at the 3′ end thatserves to determine the site from which DNA polymerase startsamplification satisfies the aforementioned condition. Fixation of thebase located at the 3′ end of each primer in this manner makes itpossible to sufficiently prevent the primer set (1) from being bound to,for example, another similar isozyme gene (for example, UGT1A3 gene orUGT1A4 gene).

As described above, since the F1 primer and R1 primer each can be anyprimer as long as the base located at the 3′ end is fixed, the lengthitself of each primer is not particularly limited and can be adjustedsuitably to be common length. The length of the primers is, for example,in the range of 13- to 50-mers, preferably 14- to 45-mers, and morepreferably 15- to 40-mers. Specifically, it is preferable that the F1primer be: at least one oligonucleotide having a sequence identical tothat of a region extending from adenine (A) at base 2120 to beconsidered as the first base to any one of the 18^(th) to 22^(nd) bases(preferably the 19^(th) to 22^(nd) bases and more preferably the 19^(th)to 21^(st) bases) in the direction toward the 5′ end in the basesequence of SEQ ID NO: 1; or at least one oligonucleotide having asequence identical to that of a region extending from cytosine (C) atbase 2140 to be considered as the first base to any one of the 18^(th)to 34^(th) bases (preferably the 21^(st) to 33^(rd) bases and morepreferably the 24^(th) to 31^(st) bases) in the direction toward the 5′end in the base sequence of SEQ ID NO: 1. Furthermore, it is preferablethat the R1 primer be: at least one oligonucleotide complementary to aregion extending from guanine (G) at base 2226 to be considered as thefirst base to any one of the 17^(th) to 27^(th) bases (preferably the19^(th) to 26^(th) bases and more preferably the 19^(th) to 23^(rd)bases) in the direction toward the 3′ end in the base sequence of SEQ IDNO: 1; or at least one oligonucleotide complementary to a regionextending from cytosine (C) at base 2198 to be considered as the firstbase to any one of the 22^(nd) to 39^(th) bases (preferably the 23^(rd)to 36^(th) bases and more preferably the 25^(th) to 34^(th) bases) inthe direction toward the 3′ end in the base sequence of SEQ ID NO: 1.Since each 3′ end of the F1 primer and the R1 primer is fixed, theregion to be elongated from the primer is, for example, one of a regionfrom base 2121 to base 2225, a region from base 2141 to base 2197, aregion from base 2121 to base 2197, and a region from base 2141 to base2225 in SEQ ID NO: 1 as described above. However, the length of thewhole amplification product obtained varies according to the length ofthe primer to be used.

Furthermore, it is not necessary for the R1 primer and the F1 primer tobe oligonucleotides perfectly complementary to the base sequenceindicated in SEQ ID NO: 1 and to the strand complementary to the basesequence, respectively. In other words, the part excluding the baselocated at the 3′ end in each primer may be different in one to fivebases from that of a perfectly complementary oligonucleotide.

Specific examples of the F1 primer and the R1 primer are indicated belowbut the present invention is not limited thereto. The combination ofthese F1 primer and R1 primer is not limited by any means. Specifically,however, a primer set (1′) is particularly preferable, which includes aF1′ primer composed of oligonucleotide of SEQ ID NO: 4 or SEQ ID NO: 81,and a R1′ primer composed of oligonucleotide of SEQ ID NO: 13 or SEQ IDNO: 91. “Tm (° C.)” indicated below in the table is Tm (° C.) obtainedwhen each sequence indicated below in the table was hybridized with thesequence perfectly complementary thereto. The “Tm (° C.)” is a valuecalculated by using MELTCALC software (http://www.meltcalc.com/), withparameters including an oligonucleotide concentration of 0.2 μM and asodium equivalent (Na eq.) of 50 mM (the same applies below). The Tmvalue can be calculated by using, for example, conventionally knownMELTCALC software (http://www.meltcalc.com/) or also can be determinedby the nearest neighbor method (the same applies below).

TABLE 1 Primer Sequence Tm(° C.) SEQ ID NO. F1 Primer5′-gcagcagaggggacatgaaata-3′ 56.2 2 for UGT1A1*6 5′-cagcagaggggacatgaaata-3′ 53.4 3   5′-agcagaggggacatgaaata-3′ 51.9 4   5′-gcagaggggacatgaaata-3′ 50.4 5     5′-cagaggggacatgaaata-3′ 46.7 65′-ggggacatgaaatagttgtcctagcacctgacgc-3′ 65.9 74 5′-gggacatgaaatagttgtcctagcacctgacgc-3′ 64.8 75  5′-ggacatgaaatagttgtcctagcacctgacgc-3′ 63.6 76   5′-gacatgaaatagttgtcctagcacctgacgc-3′ 62.4 77    5′-acatgaaatagttgtcctagcacctgacgc-3′ 62 78     5′-catgaaatagttgtcctagcacctgacgc-3′ 61.1 79      5′-atgaaatagttgtcctagcacctgacgc-3′ 60.4 80       5′-tgaaatagttgtcctagcacctgacgc-3′ 60.4 81        5′-gaaatagttgtcctagcacctgacgc-3′ 59.3 82         5′-aaatagttgtcctagcacctgacgc-3′ 58.7 83          5′-aatagttgtcctagcacctgacgc-3′ 58.4 84           5′-atagttgtcctagcacctgacgc-3′ 58.1 85            5′-tagttgtcctagcacctgacgc-3′ 57.9 86             5′-agttgtcctagcacctgacgc-3′ 58.3 87              5′-gttgtcctagcacctgacgc-3′ 57.2 88               5′-ttgtcctagcacctgacgc-3′ 56 89                5′-tgtcctagcacctgacgc-3′ 55.4 90 R1 Primer5′-ctcaaaaacattatgcccgagactaac-3′ 56.4 7 for UGT1A16 5′-tcaaaaacattatgcccgagactaac-3′ 55.7 8  5′-caaaaacattatgcccgagactaac-3′ 54.7 9   5′-aaaaacattatgcccgagactaac-3′ 53.5 10    5′-aaaacattatgcccgagactaac-3′ 53 11     5′-aaacattatgcccgagactaac-3′ 52.4 12      5′-aacattatgcccgagactaac-3′ 51.8 13       5′-acattatgcccgagactaac-3′ 51.1 14        5′-cattatgcccgagactaac-3′ 49.3 15         5′-attatgcccgagactaac-3′ 47.4 16          5′-ttatgcccgagactaac-3′ 46.6 175′-ccgagactaacaaaagactctttcacatcctccctttgg-3′ 65 106 5′-cgagactaacaaaagactctttcacatcctccctttgg-3′ 64 107  5′-gagactaacaaaagactctttcacatcctccctttgg-3′ 62.7 108   5′-agactaacaaaagactctttcacatcctccctttgg-3′ 62.4 109    5′-gactaacaaaagactctttcacatcctccctttgg-3′ 61.8 110     5′-actaacaaaagactctttcacatcctccctttgg-3′ 61.5 111      5′-ctaacaaaagactctttcacatcctccctttgg-3′ 60.6 112       5′-taacaaaagactctttcacatcctccctttgg-3′ 60.2 113        5′-aacaaaagactctttcacatcctccctttgg-3′ 60.6 114         5′-acaaaagactctttcacatcctccctttgg-3′ 60.4 115          5′-caaaagactctttcacatcctccctttgg-3′ 59.4 91           5′-aaaagactctttcacatcctccctttgg-3′ 58.7 116            5′-aaagactctttcacatcctccctttgg-3′ 58.4 117             5′-aagactctttcacatcctccctttgg-3′ 58.1 118              5′-agactctttcacatcctccctttgg-3′ 57.8 119               5′-gactctttcacatcctccctttgg-3′ 56.8 120                5′-actctttcacatcctccctttgg-3′ 55.9 121                 5′-ctctttcacatcctccctttgg-3′ 54.5 122

Next, as described above, the primer set (2) is a primer set of a pairof primers including a forward primer composed of the followingoligonucleotide (F2) and a reverse primer composed of the followingoligonucleotide (R2):

-   (F2): oligonucleotide that is at least one oligonucleotide having a    sequence identical to that of a region extending from guanine (G) at    base 2622 to be considered as the first base to any one of the    15^(th) to 27^(th) bases in the direction toward the 5′ end in the    UGT1A1 gene consisting of the base sequence of SEQ ID NO: 1, with    the guanine (G) being the 3′ end, and-   (R2): oligonucleotide that is at least one oligonucleotide    complementary to a region extending from cytosine (C) at base 2687    to be considered as the first base to any one of the 17^(th) to    26^(th) bases in the direction toward the 3′ end in the UGT1A1 gene    consisting of the base sequence of SEQ ID NO: 1, with guanine (G)    complementary to the cytosine (C) at base 2687 being the 3′ end.

The primer set (2) is a primer set for amplifying a DNA strand includinga region from base 2623 to base 2686 in SEQ ID NO: 1 as well as a strandcomplementary thereto. Base 2635 in this region (i.e. base 2635 in SEQID NO: 1) is known for the presence of a point mutation (2635C, 2635A)that affects the function of UGT1A1, and the polymorphism thereof isUGT1A1*27 described above. In the present invention, the polymorphism ofthis site can be indicated as 2635C/C or 26359A/A in the case ofhomozygote and as 2635C/A in the case of heterozygote. Hereinafter, thisprimer set (2) also may be referred to as a “primer set for UGT1A1*27”.When only the polymorphism UGT1A1*27 is to be analyzed, it is sufficientto use only the primer set for UGT1A1*27.

For the same reason as that described with respect to the primer set(1), in the present invention, the F2 primer and the R2 primer of theprimer set (2) can be any primers, as long as the base located at the 3′end that serves to determine the site from which DNA polymerase startsamplification satisfies the aforementioned condition. Accordingly, thelength itself of the F2 primer and the R2 primer is not particularlylimited and can be, for example, as described above. Specifically, it ispreferable that the F2 primer be at least one oligonucleotide having asequence identical to that of a region extending from guanine (G) atbase 2622 to be considered as the first base to any one of the 15^(th)to 27^(th) bases (preferably the 16^(th) to 27^(th) bases and morepreferably the 17^(th) to 27^(th) bases) in the direction toward the 5′end in the base sequence of SEQ ID NO: 1. Furthermore, it is preferablethat the R2 primer be at least one oligonucleotide complementary to aregion extending from cytosine (C) at base 2687 to be considered as thefirst base to any one of the 17^(th) to 26^(th) bases (preferably the18^(th) to 26^(th) bases and more preferably the 19^(th) to 26^(th)bases) in the direction toward the 3′ end in the base sequence of SEQ IDNO: 1. Since each 3′ end of the F2 primer and the R2 primer is fixed,the region to be elongated from the primer is, for example, a regionfrom base 2623 to base 2686 in SEQ ID NO: 1 as described above. However,the length of the whole amplification product obtained varies accordingto the length of the primer to be used.

Furthermore, it is not necessary for the R2 primer and the F2 primer tobe oligonucleotides perfectly complementary to the base sequenceindicated in SEQ ID NO: 1 and to the strand complementary to the basesequence, respectively. In other words, the part excluding the baselocated at the 3′ end in each primer may be different in one to fivebases from that of a perfectly complementary oligonucleotide.

Specific examples of the F2 primer and the R2 primer are indicated belowbut the present invention is not limited thereto. The combination ofthese F2 primer and R2 primer is not limited by any means. Specifically,however, a primer set (2′) is particularly preferable, which includes aF2′ primer composed of oligonucleotide of SEQ ID NO: 21 or SEQ ID NO:92, and a R2′ primer composed of oligonucleotide of SEQ ID NO: 29 or SEQID NO: 98.

TABLE 2 Primer Sequence Tm(° C.) SEQ ID NO. F2 Primer5′-ccttttcacagaactttctgtgcgacg-3′ 60.5 92 for UGT1A1*27 5′-cttttcacagaactttctgtgcgacg-3′ 58.9 93  5′-ttttcacagaactttctgtgcgacg-3′ 58.3 94   5′-tttcacagaactttctgtgcgacg-3′ 58 95    5′-ttcacagaactttctgtgcgacg-3′ 57.7 96     5′-tcacagaactttctgtgcgacg-3′ 57.3 97      5′-cacagaactttctgtgcgacg-3′ 56.2 18       5′-acagaactttctgtgcgacg-3′ 55 19        5′-cagaactttctgtgcgacg-3′ 53.4 20         5′-agaactttctgtgcgacg-3′ 51.9 21          5′-gaactttctgtgcgacg-3′ 50.3 22           5′-aactttctgtgcgacg-3′ 48.7 23            5′-actttctgtgcgacg-3′ 47.6 24 R2 Primer5′-gccagacagatgcagagctcaatagg-3′ 60.7 98 for UGT1A1*27 5′-ccagacagatgcagagctcaatagg-3′ 58.6 99  5′-cagacagatgcagagctcaatagg-3′ 56.7 25   5′-agacagatgcagagctcaatagg-3′ 55.7 26    5′-gacagatgcagagctcaatagg-3′ 54.5 27     5′-acagatgcagagctcaatagg-3′ 53.5 28      5′-cagatgcagagctcaatagg-3′ 51.9 29       5′-agatgcagagctcaatagg-3′ 50.3 30        5′-gatgcagagctcaatagg-3′ 48.7 31         5′-atgcagagctcaatagg-3′ 47 32

Next, as described above, the primer set (3) is a primer set of a pairof primers including a forward primer composed of the followingoligonucleotide (F3) and a reverse primer composed of the followingoligonucleotide (R3):

-   (F3): oligonucleotide that is at least one oligonucleotide having a    sequence identical to that of a region extending from cytosine (C)    at base 1863 to be considered as the first base to any one of the    17^(th) to 31^(st) bases in the direction toward the 5′ end in the    UGT1A1 gene consisting of the base sequence of SEQ ID NO: 1, with    the cytosine (C) being the 3′ end, and-   (R3): oligonucleotide that is at least one oligonucleotide    complementary to a region extending from cytosine (C) at base 1928    to be considered as the first base to any one of the 16^(th) to    20^(th) bases in the direction toward the 3′ end in the UGT1A1 gene    consisting of the base sequence of SEQ ID NO: 1, with guanine (G)    complementary to the cytosine (C) at base 1928 being the 3′ end.

The primer set (3) is a primer set for amplifying a DNA strand includinga region from base 1864 to base 1927 in SEQ ID NO: 2 as well as a strandcomplementary thereto. In this region, a mutation (six repeated TAs,seven repeated TAs) in the TATA box starting at base 1895 of SEQ ID NO:1 is known as a point mutation that affects the function of UGT1A1, andthe polymorphism thereof is UGT1A1*28 described above. In the presentinvention, the polymorphism of this site can be indicated as1895-TA7/TA7 or 1895-TA6/TA6 in the case of homozygote and as1859-TA6/TA7 in the case of heterozygote. SEQ ID NO: 1 is a sequence ofthe polymorphism UGT1A1*28 having seven repeated TAs. Hereinafter, thisprimer set (3) also may be referred to as a “primer set for UGT1A1*28”.When only the polymorphism UGT1A1*28 is to be analyzed, it is sufficientto use only the primer set for UGT1A1*28.

In the present invention, from the same reason as that described withrespect to the primer set (1), the F3 primer and the R3 primer of theprimer set (3) can be any primers as long as the base located at the 3′end that serves to determine the site from which DNA polymerase startsamplification satisfies the aforementioned condition. Accordingly, thelength itself of the F3 primer and the R3 primer is not particularlylimited and can be, for example, as described above. Specifically, it ispreferable that the F3 primer be at least one oligonucleotide having asequence identical to that of a region extending from cytosine (C) atbase 1863 to be considered as the first base to any one of the 17^(th)to 31^(st) bases (preferably the 18^(th) to 28^(th) bases and morepreferably the 19^(th) to 24^(th) bases) in the direction toward the 5′end in the base sequence of SEQ ID NO: 1. Furthermore, it is preferablethat the R3 primer be at least one oligonucleotide complementary to aregion extending from cytosine (C) at base 1928 to be considered as thefirst base to any one of the 16^(th) to 20^(th) bases (preferably the17^(th) to 20^(th) bases and more preferably the 17^(th) to 19^(th)bases) in the direction toward the 3′ end in the base sequence of SEQ IDNO: 1. Since each 3′ end of the F3 primer and the R3 primer is fixed,the region to be elongated from the primer is, for example, a regionfrom base 1864 to base 1927 in SEQ ID NO: 2 as described above. However,the length of the whole amplification product obtained varies accordingto the length of the primer to be used.

Furthermore, it is not necessary for the R3 primer and the F3 primer tobe oligonucleotides perfectly complementary to the base sequenceindicated in SEQ ID NO: 1 and to the strand complementary to the basesequence, respectively. In other words, the part excluding the baselocated at the 3′ end in each primer may be different in one to fivebases from that of a perfectly complementary oligonucleotide.

Specific examples of the F3 primer and the R3 primer are indicated belowbut the present invention is not limited thereto. The combination ofthese F3 primer and R3 primer is not limited by any means. Specifically,however, a primer set (3′) is particularly preferable, which includes aF2′ primer composed of oligonucleotide of SEQ ID NO: 42 or SEQ ID NO:123 and a R3′ primer composed of oligonucleotide of SEQ ID NO: 4(1 orSEQ ID NO: 48.

TABLE 3 Primer Sequence Tm(° C.) SEQ ID NO. F3 Primer5′-agctttttatagtcacgtgacacagtcaaac-3′ 59.5 123 for UGT1A1*28 5′-gctttttatagtcacgtgacacagtcaaac-3′ 58.7 124  5′-ctttttatagtcacgtgacacagtcaaac-3′ 56.8 33   5′-tttttatagtcacgtgacacagtcaaac-3′ 56.1 34    5′-ttttatagtcacgtgacacagtcaaac-3′ 55.8 35     5′-tttatagtcacgtgacacagtcaaac-3′ 55.4 36      5′-ttatagtcacgtgacacagtcaaac-3′ 55 37       5′-tatagtcacgtgacacagtcaaac-3′ 54.5 38        5′-atagtcacgtgacacagtcaaac-3′ 54.8 39         5′-tagtcacgtgacacagtcaaac-3′ 54.5 40          5′-agtcacgtgacacagtcaaac-3′ 54.8 41           5′-gtcacgtgacacagtcaaac-3′ 53.5 42            5′-tcacgtgacacagtcaaac-3′ 52.1 43             5′-cacgtgacacagtcaaac-3′ 50.6 44              5′-acgtgacacagtcaaac-3′ 48.7 45 R3 Primer5′-cgcctttgctcctgccagag-3′ 59.9 46 for UGT1A1*28 5′-gcctttgctcctgccagag-3′ 57.3 47   5′-cctttgctcctgccagag-3′ 54.1 48   5′-ctttgctcctgccagag-3′ 51.3 49     5′-tttgctcctgccagag-3′ 49.7 50

Furthermore, each primer of the aforementioned primer sets (1) to (3)may be, for example, one with the 5′ end to which any conventionallyknown sequence has been added in order to increase the amplificationreaction temperature.

Preferably, a primer set for amplifying the UGT1A1 gene of the presentinvention including at least one of the aforementioned primer sets (1)to (3) is used, for example, in amplifying the UGT1A1 gene in abiological sample such as a whole blood sample. Particularly, when theprimer set for amplifying the UGT1A1 gene of the present invention isused in combination with a probe for detecting a polymorphism asdescribed later, it is preferable that the ratio of the whole bloodsample to be added to the reaction solution for amplifying a gene be 0.1to 0.5 vol %. This will be described later.

<Reagent for Amplifying UGT1A1 Gene>

As described above, a reagent for amplifying the UGT1A1 gene of thepresent invention is a reagent for amplifying the UGT1A1 gene by a geneamplification method, wherein the reagent includes a primer set foramplifying the UGT1A1 gene of the present invention. The reagent foramplifying the UGT1A1 gene of the present invention is characterized byincluding a primer set of the present invention and, for example,components other than this are not limited by any means.

For example, in order to detect an amplification product obtained by agene amplification method in which a primer set of the present inventionis used, the reagent for amplifying the UGT1A1 gene of the presentinvention further may include a probe that can hybridize to a site to bedetected in the UGT1A1 gene. As described above, the primer set foramplifying the UGT1A1 gene of the present invention allows amplificationproducts of one to three target regions in the UGT1A1 gene to beobtained by a gene amplification method according to, for example, thetype of the primer sets (1) to (3) included therein. Accordingly, when aprobe complementary to the sequence to be detected in each target regiondescribed above is allowed to coexist, for example, the presence orabsence of amplification or the genotype (polymorphism) of the site tobe detected can be detected by the method described later. Such probesand the method of using them are explained later in the description ofthe polymorphism analysis method. Furthermore, it is preferable that thereagent for amplifying the UGT1A1 gene of the present invention be usedin amplifying the UGT1A1 gene in a biological sample such as wholeblood. Particularly, when the reagent for amplifying the UGT1A1 gene ofthe present invention is used in combination with the probe describedabove, it is preferable that the ratio of the whole blood sample to beadded to the reaction solution for amplifying a gene be 0.1 to 0.5 vol%. In the present invention, the term “sequence to be detected” denotesa sequence including a site (site to be detected) at which apolymorphism is generated.

The form of the reagent for amplifying the UGT1A1 gene of the presentinvention is not particularly limited and it may be, for example, aliquid reagent containing a primer set for amplifying the UGT1A1 gene ofthe present invention or a dry reagent that is to be suspended in asolvent before use. Furthermore, the content of the primer set foramplifying the UGT1A1 gene also is not particularly limited.

<Method of Manufacturing Amplification Product>

As described above, the method of manufacturing an amplification productof the present invention is a method of manufacturing an amplificationproduct of the UGT1A1 gene by a gene amplification method, wherein themethod includes the following step (I):

-   (I) amplifying the UGT1A1 gene in a reaction solution using a primer    set for amplifying the UGT1A1 gene of the present invention, with    nucleic acid contained in a sample being used as a template.

When a primer set for amplifying the UGT1A1 gene of the presentinvention is used to perform an amplification reaction in this manner,the target region of the UGT1A1 gene can be amplified as describedabove. Furthermore, when the primer set for amplifying the UGT1A1 geneof the present invention includes two of the primer sets (1) to (3), twotarget regions including two sites to be detected, respectively, in theUGT1A1 gene can be amplified simultaneously in the same reactionsolution. Moreover, when the primer set for amplifying the UGT1A1 geneof the present invention includes all the primer sets (1) to (3), threetarget regions including three sites to be detected, respectively, inthe UGT1A1 gene can be amplified simultaneously in the same reactionsolution. The target regions to be amplified according to the presentinvention are regions including the sites to be detected at whichrespective polymorphisms (UGT1A1*28, UGT1A1*6, and UGT1A1*27) aregenerated, respectively, as described above. The method of manufacturingan amplification product of the present invention is characterized inthat a primer set of the present invention is used, and, for example,the type of and conditions for the gene amplification method are notlimited by any means.

The gene amplification method is not particularly limited as describedabove. Examples thereof include the polymerase chain reaction (PCR)method, a nucleic acid sequence based amplification (NASBA) method, atranscription-mediated amplification (TMA) method, and a stranddisplacement amplification (SDA) method. Particularly, the PCR method ispreferable. The present invention is described below using the PCRmethod as an example but is not limited thereby.

The sample to which the present invention is to be applied is notparticularly limited as long as it contains, for example, nucleic acidto serve as a template. However, it is preferable that the presentinvention be applied to, for example, a contaminated sample. Examples ofthe contaminated sample include whole blood, cells in the mouth (forexample, oral mucosa), somatic cells of nails and hairs, germ cells,expectoration, amniotic fluid, paraffin-embedded tissue, urine, gastricjuice (for example, gastric lavage fluid), and suspensions thereof.According to the method of manufacturing an amplification product usinga primer set of the present invention, for example, even in the case ofa sample (particularly, a biological sample such as whole blood or cellsin the mouth) with various contaminants, the method is less subject tothe effect thereof and allows the target region in the UGT1A1 gene to beamplified specifically. Thus, according to the present invention, even ahighly contaminated sample, which is difficult to use in theconventional methods, can be used as it is, for instance, without beingpretreated, for example, without being purified. Therefore, it can besaid that an amplification product can be prepared quicker as comparedto the conventional method also from the viewpoint of the pretreatmentof the sample.

The ratio of the sample to be added to the reaction solution is notparticularly limited. Specifically, when the sample is a biologicalsample (for example, a whole blood sample), the lower limit of the ratiothereof to be added to the reaction solution is, for example, preferablyat least 0.01 vol %, more preferably at least 0.05 vol %, and furtherpreferably at least 0.1 vol %. Furthermore, the upper limit of the ratiothereof to be added also is not particularly limited and is, forexample, preferably 2 vol % or lower, more preferably 1 vol % or lower,and further preferably 0.5 vol % or lower.

When an optical detection to be described later is intended to beperformed, particularly, when an optical detection is performed using alabeled probe, it is preferable that the ratio of a biological sample,such as a whole blood sample, to be added to the reaction solution beset at, for example, 0.1 to 0.5 vol %. Generally, in the PCR reaction, aheat treatment is carried out to denature DNA (i.e. to dissociate itinto a single-stranded DNA). This heat treatment may denature, forexample, sugar or protein contained in the sample and thereby maygenerate an insolubilized precipitate or turbidity. Therefore, when thepresence or absence of an amplification product or the genotype(polymorphism) of a site to be detected is to be checked by an opticalmethod, the generation of such a precipitate or turbidity may affect themeasurement accuracy. However, when the ratio of the whole blood sampleto be added to the reaction solution is set in the range describedabove, for example, an effect caused by generation of, for example, aprecipitate due to denaturation can be prevented sufficiently andthereby the accuracy of measurement carried out by the optical methodcan be improved, although the mechanism thereof is unknown. Furthermore,since it also can sufficiently prevent PCR from being inhibited due tothe contaminants contained in a whole blood sample, the amplificationefficiency can be improved further. Accordingly, when in addition to theuse of a primer set of the present invention, the ratio of the samplesuch as a whole blood sample to be added is set in the aforementionedrange, the need to pretreat the sample additionally can be omitted.

Furthermore, the ratio of the whole blood sample in the reactionsolution can be indicated not in the aforementioned volume ratio (forexample, 0.1 to 0.5 vol %) but in a weight ratio of hemoglobin(hereinafter referred to as “Hb”). In this case, the ratio of the wholeblood sample in the reaction solution is, for example, preferably in therange of 0.565 to 113 g/L, more preferably in the range of 2.825 to 56.5g/L, and further preferably in the range of 5.65 to 28.25 μg/L, in termsof the amount of Hb. The ratio of the whole blood sample to be addedduring the reaction may satisfy, for example, both the volume ratio andthe Hb weight ratio, or one of them.

The whole blood may be any one of, for example, hemolyzed whole blood,unhemolyzed whole blood, anticoagulated whole blood, and whole bloodcontaining coagulated fractions.

In the present invention, the target nucleic acid contained in a sampleis, for example, DNA. The aforementioned DNA may be DNA containedoriginally in the sample, such as a biological sample, or anamplification product DNA obtained through amplification by a geneamplification method. In the latter case, an example thereof is cDNAthat is generated from RNA (for example, total RNA or mRNA) containedoriginally in the sample by a reverse transcription reaction (forinstance, reverse transcription PCR (RT-PCR)).

In the method of manufacturing an amplification product of the presentinvention, it is preferable that albumin further be added to thereaction solution before the start of a gene amplification reaction.Such addition of albumin further can reduce the effect of generation ofa precipitate or turbidity described above and also further can improvethe amplification efficiency. Specifically, it is preferable thatalbumin be added before the amplification reaction in step (I) or a stepof dissociation into a single-stranded DNA.

The ratio of albumin to be added to the reaction solution is, forexample, in the range of 0.01 to 2 wt %, preferably 0.1 to 1 wt %, andmore preferably 0.2 to 0.8 wt %. The albumin is not particularlylimited. Examples thereof include bovine serum albumin (BSA), humanserum albumin, rat serum albumin, and horse serum albumin. One of themmay be used or two or more of them may be used in combination.

Next, a method of manufacturing an amplification product of the presentinvention is described using an example in which with respect to a wholeblood sample including DNA as target nucleic acid, amplificationproducts of three target regions of the UGT1A1 gene are produced by PCRusing primer sets for amplifying the UGT1A1 gene of the presentinvention including the aforementioned primer sets (1) to (3). Thepresent invention is characterized by using primer sets of the presentinvention and other configurations and conditions are not limited by anymeans.

First, a PCR reaction solution is prepared. The ratio of the primer setsof the present invention to be added is not particularly limited.However, it is preferable that F primers of the primer sets (1) to (3)each be added to he 0.1 to 2 μmol/L, more preferably 0.25 to 1.5 μmol/L,and particularly preferably 0.5 to 1 μmol/L. Furthermore, it ispreferable that R primers of the primer sets (1) to (3) each be added tobe 0.1 to 2 μmol/L, more preferably 0.25 to 1.5 μmol/L, and particularlypreferably 0.5 to 1 μmol/L. The ratio (F:R, molar ratio) between the Fprimer and the R primer to be added to each primer set is notparticularly limited. It is, for example, preferably 1:0.25 to 1:4 andmore preferably 1:0.5 to 1:2.

The ratio of the whole blood sample in the reaction solution is notparticularly limited but is preferably in the range described above. Thewhole blood sample may be added to the reaction solution without beingtreated or may be added to the reaction solution after being dilutedwith a solvent such as water or a buffer solution beforehand. When thewhole blood sample is diluted beforehand, the dilution ratio is notparticularly limited. It can be set so that, for example, the finalratio of the whole blood added to the reaction solution is in theaforementioned range, for example, 1:100 to 1:2000 and preferably 1:200to 1:1000.

Other composition components in the reaction solution are notparticularly limited and can be conventionally known components, whoseratios also are not particularly limited. Examples of the compositioncomponents include DNA polymerase, nucleotide (nucleoside triphosphate(dNTP)), and a solvent. Furthermore, as described above, it ispreferable that the reaction solution further contain albumin. In thereaction solution, the order of addition of the respective compositioncomponents is not limited by any means.

The DNA polymerase is not particularly limited and, for example, aconventionally known thermoduric bacteria-derived polymerase can beused. Specifically, for example, Thermus aquaticus-derived DNApolymerase (U.S. Pat. Nos. 4,889,818 and 5,079,352) (trade name: Taqpolymerase), Thermus thermophilus-derived DNA polymerase (WO 91/09950)(rTth DNA polymerase), Pyrococcus furiosus-derived DNA polymerase (WO9219688) (Pfu DNA polymerase; manufactured by Stratagenes), andThermococcus litoralis-derived DNA polymerase (EP-A 455 430) (Trademark:Vent; manufactured by Biolab New England) are commercially available.Particularly, Thermus aquaticus-derived thermostable DNA polymerase ispreferable.

The ratio of DNA polymerase to he added to the reaction solution is notparticularly limited and is, for example, 1 to 100 U/mL, preferably 5 to50 U/mL, and more preferably 20 to 30 U/mL. With respect to the unit ofactivity (U) of DNA polymerase, generally, 1 U denotes the activity thatallows all 10 nmol of nucleotide to be taken into an acid-insolubleprecipitate in 30 minutes at 74° C. in a reaction solution for activitymeasurement, with an activated salmon sperm DNA being used as a templateprimer. The composition of the reaction solution for activitymeasurement is, for example, 25 mM TAPS buffer (pH 9.3, 25° C.), 50 mMKCl, 2 mM MgCl₂, 1 mM mercaptoethanol, 200 μM dATP, 200 μM dGTP, 200 μMdTTP, 100 μM [α⁻³²P] dCTP, and 0.25 mg/mL activated salmon sperm DNA.

Generally, examples of the nucleoside triphosphate include dNTP (dATP,dCTP, dTTP). The ratio of dNTP to be added to the reaction solution isnot particularly limited and is, for example, 0.01 to 1 mmol/L,preferably 0.05 to 0.5 mmol/L, and more preferably 0.1 to 0.3 mmol/L.

Examples of the solvent include buffer solutions such as Tris-HCl,Tricine, MES, MOPS, HEPES, and CAPS. Commercially available PCR buffersolutions or buffer solutions of commercially available PCR kits can beused.

Furthermore, the PCR reaction solution further may contain heparin,betaine, KCl, MgCl₂, MgSO₄, glycerol, etc. The ratios thereof to beadded can be set in ranges in which the PCR reaction is not inhibited.

The total volume of the reaction solution is not particularly limitedand can be determined suitably according to, for example, the equipment(thermal cycler) to be used. It is generally 1 to 500 μL and preferably10 to 100 μL.

Subsequently, PCR is performed. The cycle conditions in PCR are notparticularly limited, and, for example, (1) dissociation of wholeblood-derived double-stranded DNA into single-stranded DNA, (2)annealing of a primer, and (3) elongation of a primer (polymerasereaction) are as described below. Furthermore, the number of cycles alsois not particularly limited but preferably is at least 30, with thefollowing three steps (1) to (3) being considered as one cycle. Theupper limit thereof, in total, is not particularly limited and, forexample, is 100 cycles or less, preferably 70 cycles or less, andfurther preferably 50 cycles or less. The change in temperature in eachstep can be controlled automatically using, for example, a thermalcycler. When primer sets of the present invention are used, since theyare excellent in amplification efficiency as described above, 50 cyclescan be completed in approximately one hour (preferably within one hour)according to the present invention, while it takes approximately threehours to complete 50 cycles according to the conventional methods.

TABLE 4 Temperature (° C.) and Time (sec) (1) Dissociation of single-For example, 90 to 99° C., 1 to 120 sec stranded DNA Preferably, 92 to95° C., 1 to 60 sec (2) Annealing of primer For example, 40 to 70° C., 1to 300 sec Preferably, 50 to 70° C., 5 to 60 sec (3) Elongation reactionFor example, 50 to 80° C., 1 to 300 sec Preferably, 50 to 75° C., 5 to60 sec

In the manner described above, amplification products complementary tothe three target regions in the UGT1A1 gene can be produced. When anamplification product complementary to one or those complementary to twoof the three target regions are to be produced, a primer set foramplifying the UGT1A1 gene of the present invention containing one ortwo of the primer sets (1) to (3) corresponding to the target region(s)can he used.

The method of manufacturing an amplification product of the presentinvention further may include a step of detecting an amplificationproduct of a target region obtained by the aforementioned amplificationreaction. This makes it possible to detect the presence or absence ofthe amplification product or the genotype (polymorphism, UGT1A1*6,UGT1A1*27, or UGT1A1*28) in the target region in the UGT1A1 gene. Thepresence or absence of the amplification product can be checked by aconventionally known method. Specifically, it can be checked by, forexample, further adding a probe (for instance, a fluorescently-labeledprobe) that can hybridize to a site to be detected in the UGT1A1 gene tothe reaction solution in step (I), and further in step (II), measuringthe fluorescence intensity of the fluorescent label in the probe withrespect to the reaction solution. Furthermore, when two or three targetregions are to be amplified, it can be checked by, for example, furtheradding two or three probes (for instance, fluorescently-labeled probes)that can hybridize to the respective sites to be detected in the UGT1A1gene to the reaction solution in step (I), and further in step (II),measuring the fluorescence intensity of the fluorescent label in eachprobe with respect to the reaction solution. Detection of polymorphisms,UGT1A1*6, UGT1A1*27, and UGT1A1*28, in the UGT1A1 gene is describedbelow as an embodiment of the present invention.

<UGT1A1 Gene Polymorphism Analysis Method>

A UGT1A1 gene polymorphism analysis method of the present invention is amethod of analyzing the polymorphism of a site to be detected in theUGT1A1 gene, wherein the method includes the following steps (i) to(iv):

(i) amplifying a region including a site to be detected in the UGT1A1gene in a reaction solution by a method of manufacturing anamplification product according to the present invention,

(ii) preparing a reaction solution that contains the amplificationproduct obtained in step (i) and a probe capable of hybridizing to thesite to be detected,

(iii) measuring signal values that indicate melting states of ahybridization product between the amplification product and the probewhile changing the temperature of the reaction solution, and

(iv) determining a polymorphism of the site to be detected from a changein the signal values accompanying a change in the temperature.

In this manner, when an amplification product is produced using a primerset for amplifying the UGT1A1 gene of the present invention, it ispossible to amplify the target region including a site to be detected ofa polymorphism (UGT1A1*6, UGT1A1*27, or UGT1A1*28) in the UGT1A1 gene asdescribed above and to analyze the polymorphism of the site to bedetected in the target region.

The probe to be used in step (ii) is not particularly limited. Examplesthereof include a probe that hybridizes to the site where thepolymorphism UGT1A1*6 is generated (hereinafter, also referred to as a“probe for UGT1A1*6”), a probe that hybridizes to the site where thepolymorphism UGT1A1*27 is generated (hereinafter, also referred to as a“probe for UGT1A1*27”), and a probe that hybridizes to the site wherethe polymorphism UGT1A1*28 is generated (hereinafter, also referred toas a “probe for UGT1A1*28”). Preferably, these probes each are a probecomplementary to a sequence to be detected containing the aforementionedsequence to be detected. Any one of those probes may be used or two orall three of them may be used This can be determined, for example,according to the type of the target region(s) amplified with a primerset for amplifying the UGT1A1 gene of the present invention. When two orthree probes are used, for example, the polymorphisms of two sites to bedetected or all the three sites to be detected can he analyzed using thesame reaction solution.

The probes for detecting the polymorphisms are not particularly limitedand can be configured by a conventionally known method. For instance,they each may be designed as a sequence to be detected containing a siteto be detected of a polymorphism, based on the sequence of a sensestrand or the sequence of an antisense strand of the UGT1A1 gene.Furthermore, the base located at the site to be detected of apolymorphism can be determined suitably according to the type of eachpolymorphism. In other words, in the case of UGT1A1*6, since thepolymorphisms of “G” and “A” at base 2160 in SEQ ID NO: 1 have beenknown, examples of the probe include a probe complementary to either asequence to be detected including G at base 2160 or a sequence to bedetected including A at base 2160 (a probe for detecting a sensestrand), and a probe complementary to a sequence of an antisense strandthereof (a probe for detecting an antisense strand). Furthermore, in thecase of UGT1A1*27, since the polymorphisms of “C” and “A” at base 2635in SEQ ID NO: 1 have been known, examples of the probe include a probecomplementary to either a sequence to be detected including C at base2635 or a sequence to be detected including A at base 2635 (a probe fordetecting a sense strand), and a probe complementary to a sequence of anantisense strand thereof (a probe for detecting an antisense strand).Moreover, in the case of UGT1A1*28, since the polymorphisms of “sixrepeated TAs: TA6” and “seven repeated TAs: TA7” have been known in thebases of the TATA box starting at base 1895 in SEQ ID NO: 1, examples ofthe probe include a probe complementary to either a sequence to bedetected including six TAs repeated from base 1895 or a sequence to bedetected including seven TAs repeated from base 1895 (a probe fordetecting a sense strand), and a probe complementary to a sequence of anantisense strand thereof (a probe for detecting an antisense strand). Asdescribed above, when a probe is designed, with the base located at thesite to be detected where a polymorphism is generated being set to beany one of the bases as described above, it is also possible to judgewhat type of polymorphism is expressed at each site to be detected in anUGT1A1 gene by the method as described later.

The probe can be added to an amplified reaction solution after step (i)i.e. after a target region in the UGT1A1 gene is subjected to anamplification reaction. However, it is preferable that the probe beadded to a reaction solution before the amplification reaction in step(i) since this allows analysis to be performed easily and quickly.

The ratio of the probe to be added to the reaction solution is notparticularly limited. For example, each probe is added to be preferablyin the range of 10 to 400 nmol and more preferably in the range of 20 to200 nmol. When a fluorescent dye is used as the label for a probe, anunlabeled probe with a sequence identical to that of the labeled probemay be used in combination with the labeled probe, for example, in orderto adjust the fluorescence intensity to be detected, and the unlabeledprobe may include a phosphate group added to the 3′ end thereof. In thiscase, the molar ratio between the labeled probe and the unlabeled probeis preferably, for example, 1:10 to 10:1. The length of the probe is notparticularly limited. It is, for example, 5- to 50-mers and preferably10- to 30-mers.

The Tm value is described. When a solution containing double-strandedDNA is heated, the absorbance at 260 nm increases. This is becauseheating releases the hydrogen bonds between both strands in thedouble-stranded DNA to dissociate it into single-stranded DNA (i.e. DNAmelting). When all double-stranded DNAs are dissociated intosingle-stranded DNAs, the absorbance thereof indicates approximately 1.5times that obtained at the start of heating (i.e. absorbance of onlydouble-stranded DNAs), which makes it possible to judge that melting iscompleted thereby. Based on this phenomenon, the melting temperature Tmgenerally is defined as a temperature at which the absorbance hasreached 50% of the total increase in absorbance.

In the aforementioned step the measurement of the signal values thatindicate the melting states of the hybridization product between theamplification product and the probe may be a measurement of absorbanceat 260 nm as described above but may be a measurement of the signal of alabeling substance. Specifically, it is preferable that a labeled probelabeled with a labeling substance be used as the aforementioned probe toperform the measurement of the signal of the labeling substance. Thelabeled probe can be, for example, a labeled probe that exhibits asignal independently but does not exhibit a signal after hybridization,or a labeled probe that does not exhibit a signal independently butexhibits a signal after hybridization. The former probe does not exhibita signal after forming a hybrid (double-stranded DNA) with a sequence tobe detected but exhibits a signal when the probe is released by heating.On the other hand, the latter probe exhibits a signal after forming ahybrid (double-stranded DNA) with a sequence to be detected but thesignal is reduced (quenched) when the probe is released by heating.Accordingly, when the signal exhibited by the label is detected under acondition (absorption wavelength etc.) specific to the signal, theprogress of melting of the hybridization product and the Tm value can bedetermined as in the case of the measurement of absorbance at 260 nm.

In the present invention, as described above, it is also possible tocheck polymorphisms with respect to amplification products of two orthree target regions amplified in the same reaction solution.Accordingly, when two or three types of probes are used, it ispreferable that they be labeled with different labels each of which isdetected under its own condition. The use of different labels asdescribed above makes it possible to analyze each amplification productseparately by changing the detection conditions even in the samereaction solution.

Specific examples of labeling substances in the labeled probes include afluorescent dye (fluorophore). A specific example of the labeled probesis preferably a probe that, for example, has been labeled with afluorescent dye, exhibits fluorescence independently, and allowsfluorescence to be reduced (for example, quenched) after hybridization.Generally, a probe that utilizes such a fluorescence quenchingphenomenon is referred to as a fluorescence quenching probe.Particularly, with respect to the aforementioned probe, it is preferablethat the 3′ end or 5′ end of oligonucleotide be labeled with afluorescent dye and the base located at the end to be labeled be C. Inthis case, in the sequence to be detected, to which the labeled probehybridizes, it is preferable that the base sequence of the labeled probebe designed so that the base to be paired with the end base C of thelabeled probe or the base located 1 to 3 bases apart from the base to bepaired be G. Generally, such a probe is referred to as a guaninequenching probe and is known as so-called QProbe (registered trademark).When such a guanine quenching probe hybridizes to a sequence to bedetected, C located at the end, which has been labeled with afluorescent dye, approaches G in the DNA to be detected, and thereby aphenomenon occurs in which the emission of the fluorescent dye decreases(the fluorescence intensity decreases). The use of such a probe makes itpossible to verify hybridization and dissociation easily according to achange in the signal.

The fluorescent dye is not particularly limited. Examples thereofinclude fluorescein, phosphor, rhodamine, and polymethine dyederivative. Examples of commercially available fluorescent dye includeBODIPY FL (brand name, manufactured by Molecular Probe Inc.),FluorePrime (trade name, manufactured by Amersham Pharmacia), Fluoredite(trade name, manufactured by Millipore Corporation), FAM (manufacturedby ABI), Cy3 and Cy5 (manufactured by Amersham Pharmacia), and TAMRA(manufactured by Molecular Probe Inc.). The combination of fluorescentdyes to be used for a plurality of probes is not particularly limited aslong as, for example, it allows the respective probes to be detectedunder different conditions. Examples thereof include a combination ofPacific Blue (with a detection wavelength is 450 to 480 nm), TAMRA (witha detection wavelength is 585 to 700 nm), and BODIPY FL (with adetection wavelength is 515 to 555 nm).

Specific examples of the sequences of probes for detecting thepolymorphisms, UGT1A1*6, UGT1A1*27, and UGT1A1*28, are indicated below,but the present invention is not limited thereto. The following probe(1) is an example of the probe for UGT1A1*6 and is a probe for detectingan antisense strand. The following probe (2) is an example of the probefor UGT1A1*27, the following probe (2-1) is a probe for detecting anantisense strand and the following probe (2-2) is a probe for detectinga sense strand. Furthermore, the following probe (3) is an example ofthe probe for UGT1A1*28 and is a probe for detecting an antisensestrand.

Probe (1)

Oligonucleotide that is at least one oligonucleotide having a sequenceidentical to that of a region extending from cytosine (C) at base 2173to be considered as the first base to any one of the 17^(th) to 25^(th)bases in the direction toward the 5′ end in SEQ ID NO: 1, with thecytosine being the 3′ end.

Probe (2)

At least one oligonucleotide selected from:

-   (2-1) Oligonucleotide that is at least one oligonucleotide having a    sequence identical to that of a region extending from cytosine (C)    at base 2645 to be considered as the first base to any one of the    15^(th) to 22^(nd) bases in the direction toward the 5′ end in SEQ    ID NO: 1, with the cytosine being the 3′ end, and-   (2-2) Oligonucleotide that is at least one oligonucleotide    complementary to a region extending from guanine (G) at base 2625 to    be considered as the first base to any one of the 17^(th) to 22^(nd)    bases in the direction toward the 3′ end in SEQ ID NO: 1, with    cytosine (C) complementary to the guanine being the 3′ end.    Probe (3)

Oligonucleotide that is at least one oligonucleotide having a sequenceidentical to that of a region extending from cytosine (C) at base 1892to be considered as the first base to any one of the 25^(th) to 31^(st)bases in the direction toward the 3′ end in SEQ ID NO: 1, with thecytosine being the 3′ end.

In the probe (1), base 2160 in SEQ ID NO: 1 is indicated with “r”, andthe “r” is G or A. In the probe (2-1), base 2635 in SEQ ID NO: 1 isindicated with “m”, and the “m” is C or A. In the probe (2-2), the basecomplementary to base 2635 in SEQ ID NO: 1 is indicated with “k”, andthe “k” is G or T. In the probe (3), the region corresponding to theTATA box starting at base 1895 in SEQ ID NO: 1 is seven repeated TAs orsix repeated TAs.

Specific examples of Probe (1), Probe (2), and Probe (3) are indicatedin the following table. “Tm(° C.)” indicated below in the table is Tm(°C.) obtained when each sequence indicated below in the table washybridized with the sequence perfectly complementary thereto. The “Tm(°C.)” is a value calculated by using MELTCALC software(http://www.meltcalc.com/), with parameters including an oligonucleotideconcentration of 0.2 μM and a sodium equivalent (Na eq.) of 50 mM.

TABLE 5 Probe Sequence Tm(° C.) SEQ ID NO. Probe (1)5′-catcagagacAgagcattttacac-3′ 53.7 51 for UGT1A1*6 5′-atcagagacAgagcattttacac-3′ 52.4 52   5′-tcagagacAgagcattttacac-3′52.1 53    5′-cagagacAgagcattttacac-3′ 50.7 54    5′-agagacAgagcattttacac-3′ 49.1 55      5′-gagacAgagcattttacac-3′47.6 56       5′-agacAgagcattttacac-3′ 45.6 57       5′-gacAgagcattttacac-3′ 44.1 58 Probe (2)5′-tggtttattcccAgtatgcaacc-3′ 53.2 59 for UGT1A1*27 5′-ggtttattcccAgtatgcaacc-3′ 50.9 60    5′-tttattcccAgtatgcaacc-3′ 49.361     5′-ttattcccAgtatgcaacc-3′ 48.5 62      5′-tattcccAgtatgcaacc-3′47.5 63       5′-attcccAgtatgcaacc-3′ 47.5 64       5′-ttcccAgtatgcaacc-3′ 46.5 65         5′-tcccAgtatgcaacc-3′ 45.466 5′-gggttgcatacTgggaataaac-3′ 53.2 100  5′-ggttgcatacTgggaataaac-3′50.9 101   5′-gttgcatacTgggaataaac-3′ 48.4 102   5′-ttgcatacTgggaataaac-3′ 46.4 103     5′-tgcatacTgggaataaac-3′ 45.5104      5′-gcatacTgggaataaac-3′ 43.2 105 Probe (3)5′-ccaTATATATATATATAtaagtaggagaggg-3′ 49.5 67 for UGT1A1*285′-ccaTATATATATATATAtaagtaggagagg-3′ 47.7 685′-ccaTATATATATATATAtaagtaggagag-3′ 45.8 695′-ccaTATATATATATATAtaagtaggaga-3′ 44.6 705′-ccaTATATATATATATAtaagtaggag-3′ 43.3 715′-ccaTATATATATATATAtaagtagga-3′ 41.9 72 5′-ccaTATATATATATATAtaagtagg-3′40.5 73

Each probe (1) indicated in the above table consists of a sequenceidentical to that of a region having A at base 2160 in SEQ ID NO: 1, andthe capitalized base indicates base 2160 in SEQ ID NO: 1. In each probe(1), the capitalized base can be replaced by “r”, and the “r” may beeither A or G. In the following table, the probes (2-1) that areexpressed with SEQ ID NOs: 59 to 66 each consist of a sequence identicalto that of a region having A at base 2635 in SEQ ID NO: 1, and thecapitalized base indicates base 2635 in SEQ ID NO: 1. In each probe(2-1), the capitalized base can be replaced by “m”, and the “m” may beeither A or C. In the following table, the probes (2-2) that areexpressed with SEQ ID NOs: 100 to 105 each consist of a sequencecomplementary to a region having A at base 2635 in SEQ ID NO: 1, and thecapitalized base indicates the base complementary to base 2635 in SEQ IDNO: 1. In each probe (2-2), the capitalized base can be replaced by “k”,and the “k” may be either T or G. Each probe (3) indicated in thefollowing table consists of a sequence identical to that of a regionhaving seven repeated TAs as the TATA box starting at base 1895 in SEQID NO: 1, and the capitalized bases indicate the seven repeated TAs. Ineach probe (3), the number of repeated TAs may be either 7 or 6. Asdescribed above, specific examples of the probe according to the presentinvention may be strands complementary to oligonucleotides indicated inthe above table.

The aforementioned probes are examples and the present invention are notlimited thereto. With respect to the probe for UGT1A1*6, a preferableprobe among the probes (1) is oligonucleotide consisting of the basesequence of SEQ ID NO: 55 or oligonucleotide consisting of the basesequence of SEQ ID NO: 56. With respect to the probe for UGT1A1*27, apreferable probe among the probes (2) is oligonucleotide consisting ofthe base sequence of SEQ ID NO: 62 or oligonucleotide consisting of thebase sequence of SEQ ID NO: 102. With respect to the probe forUGT1A1*28, a preferable probe among the probes (3) is oligonucleotideconsisting of the base sequence of SEQ ID NO: 69.

When two or more of these probes are used, as described above, it ispreferable that they be labeled with different fluorescent dyes(fluorescent dyes that are detected at different wavelengths). Forinstance, when the probes indicated in the above table are guaninequenching probes, it is preferable that in each probe for UGT1A1*6 andeach probe for UGT1A1*27, cytosine at the 3′ end thereof be labeled witha fluorescent dye (for instance, Pacific Blue or TAMRA) as describedabove and in each probe for UGT1A1*28, cytosine at the 5′ end thereof belabeled with a fluorescent dye (for instance, BODIPY FL) as describedabove. Furthermore, a probe with the 5′ end labeled with a fluorescentdye may have the 3′ end, to which a phosphate group further may beadded, in order to prevent the probe itself from elongating.

Next, with respect to the detection method of the present invention, amethod of detecting three polymorphisms, UGT1A1*28, UGT1A1 *6, andUGT1A1*27, in the UGT1A1 gene using the following probes is described asan example. However, the present invention is not limited thereto.

<Probes>

Probe for UGT1A1*6 (SEQ ID NO: 55)5′-agagacaGagcattttacac-(Pacific Blue)-3′, or (SEQ ID NO: 56)5′-gagacaGagcattttacac-(Pacific Blue)-3′ Probe for UGT1A1*27(SEQ ID NO: 62) 5′-ttattcccAgtatgcaacc-(TAMRA)-3′, or (SEQ ID NO: 102)5′-gttgcatacTgggaataaac-(TAMRA)-3′ Probe for UGT1A1*28 (SEQ ID NO: 69)5′-(BODIPY FL)-ccaTATATATATATATAtaagtaggagag-3′

First, using a reaction solution containing the aforementioned threelabeled probes added thereto, PCR was performed as described above, andthereby the three regions of the UGT1A1 gene are amplified at the sametime in the same reaction solution. The reaction solution contains, forexample, a primer set for amplifying the UGT1A1 gene of the presentinvention, DNA polymerase, dNTP, a sample containing nucleic acid toserve as a template, and the aforementioned three probes. In addition tothem, various additives that can be used for amplifying nucleic acid maybe contained.

Next, the amplification products thus obtained are dissociated and thensingle-stranded DNA obtained through dissociation is hybridized with thelabeled probes. This can be carried out through, for example, a changein the temperature of the reaction solution.

The heating temperature in the dissociation step is not particularlylimited as long as it allows the amplification products to bedissociated. It is, for example, 85 to 95° C. The heating time also isnot particularly limited and generally is 1 second to 10 minutes andpreferably 1 second to 5 minutes.

The dissociated single-stranded DNAs can be hybridized with the labeledprobes by, for example, decreasing the heating temperature employed inthe dissociation step after the dissociation step. The temperaturecondition is, for example, 40 to 50° C.

The temperature of the reaction solution is changed and thereby signalvalues that indicate the melting states of hybridization productsbetween the amplification products and the labeled probes are measured.Specifically, for example, the reaction solution (the hybridizationproducts between the single-stranded DNAs and the labeled probes) isheated, and thereby the change in the signal values accompanying thetemperature rise is measured. As described above, when, for example, aprobe (guanine quenching probe), in which the base C at the end has beenlabeled, is used, fluorescence decreases (or quenches) in the statewhere the probe has been hybridized with the single-stranded DNA, whilefluorescence is emitted in the state where the probe has beendissociated. Accordingly, for example, the hybridization product inwhich the fluorescence has decreased (or quenched) is heated graduallyand the increase in fluorescence intensity accompanying the temperaturerise may be measured.

The temperature range in which the change in fluorescence intensity isto be measured is not particularly limited. For example, the starttemperature is room temperature to 85° C. and preferably 25 to 70° C.,while the end temperature is, for example, 40 to 105° C. Furthermore,the rate of temperature rise is not particularly limited and is, forexample, 0.1 to 20° C./sec and preferably 0.3 to 5° C./sec.

Next, the Tm value is determined by analyzing a change in the signal.Specifically, the amount of change in the fluorescence intensity perunit time at each temperature (−d fluorescence intensity increase/dt) iscalculated from the fluorescence intensity obtained and the temperatureat which the lowest value is obtained is determined as the Tm value. Itis also possible to determine, as the Tm value, the point at which theamount of increase in the fluorescence intensity per unit time(fluorescence intensity increase/t) is the highest. On the contrary theamount of decrease in the fluorescence intensity is measured when thelabeled probe used is not a quenching probe but a probe that does notexhibit a signal independently but exhibits a signal afterhybridization.

In the present invention, in order to detect three polymorphisms,UGT1A1*6, UGT1A1*27, and UGT1A1*28, the respective Tm values aredetermined under conditions according to the respective labels of thethree probes. Pacific Blue, a probe for UGT1A1*6, can be detected with,for example, a detection wavelength of 450 to 480 nm, TAMRA, a probe forUGT1A1*27, with, for example, a detection wavelength of 585 to 700 nm,and BODIPY FL, a probe for UGT1A1*28, with, for example, a detectionwavelength of 515 to 555 nm.

From such Tm values, the genotypes in the respective sites to bedetected are determined. In the Tm analysis, the case of a perfectlycomplementary hybrid (match) results in a higher Tm value indicatingdissociation than that obtained in the case of a hybrid including adifferent single base (mismatch). Accordingly, when with respect to theprobe, the Tm value obtained in the case of a perfectly complementaryhybrid and the Tm value obtained in the case of a hybrid including adifferent single base are determined beforehand, the genotype at eachsite to be detected can be determined. For example, in the case wherethe base located at the site to be detected is assumed to be of amutation type (with, for instance, A at base 2160 in SEQ ID NO: 1), whenusing a probe complementary to the sequence to be detected containingthe base, the polymorphism of the amplification product can be judged asa mutation type if the Tm value of the resultant hybrid is equal to theTm value of a perfectly complementary hybrid. Furthermore, thepolymorphism of the amplification product can be judged as a wildtype(with, for example, G at base 2160 in SEQ ID NO: 1) if the Tm value ofthe resultant hybrid is equal to the Tm value of the hybrid including adifferent single base (i.e. a lower value than the Tm value of theperfectly complementary hybrid). Moreover, when both the Tm values aredetected, it can be judged as heterozygote. Thus, the genotypes of thepolymorphisms, UGT1A1*6, UGT1A1*27, and UGT1A1*28, can be judged fromthe three Tm values with respect to the respective labeled probes.

In the present invention, for example, a change in the signal duringhybridization may be measured instead by the method in which thetemperature of a reaction solution containing the probes is increased (ahybridization product is heated) and a change in the signal accompanyingthe temperature rise is measured as described above. In other words,when the temperature of the reaction solution containing theaforementioned probes is decreased to form hybridization products, thechange in the signal accompanying the temperature decrease may bemeasured.

Specifically, when using a labeled probe that exhibits a signalindependently but does not exhibit a signal after hybridization (forexample, a guanine quenching probe), the labeled probe emitsfluorescence in the state where single-stranded DNA and the probe aredissociated, but the fluorescence decreases (or quenches) when a hybridis formed through temperature decrease. Accordingly, for example, thetemperature of the reaction solution is decreased gradually and thedecrease in fluorescence intensity accompanying the temperature decreasemay be measured. On the other hand, when using a labeled probe that doesnot exhibit a signal independently but exhibits a signal afterhybridization, the labeled probe does not emit fluorescence in the statewhere single-stranded DNA and the probe are dissociated, but thefluorescence is emitted when a hybrid is formed through temperaturedecrease. Accordingly, for example, the temperature of the reactionsolution is decreased gradually and thereby the increase in fluorescenceintensity accompanying the temperature decrease may be measured.

When one or two of the three types of polymorphisms (UGT1A1*28,UGT1A1*6, and UGT1A1*27) in the UGT1A1 gene are to be analyzed, forinstance, a primer set for amplifying the UGT1A1 gene of the presentinvention may be used that includes one or two types of primer setscorresponding to the target regions that are selected from the primersets (1) to (3), and furthermore, one or two probes that hybridize totarget sites to be detected may be used.

Next, examples of the present invention are described. However, thepresent invention is not limited by the following examples.

EXAMPLE 1

Blood was collected from nine subjects using heparin lithium bloodcollection tubes (Samples 1 to 9). Subsequently, 10 μL of blood thusobtained and 90 μL of distilled water were mixed together. Further, 10μL of this mixture and 90 μL of distilled water were mixed together.Thereafter, 10 μL of the mixture was added to 40 μL of PCR reactionsolution having the following composition, and then PCR was performedusing a thermal cycler. Conditions for PCR were as follows. That is,after treating at 95° C. for 60 seconds, one cycle of treatment at 95°C. for 1 second and at 54° C. for 10 seconds was repeated for 50 cycles,and further it was treated at 95° C. for 1 second and at 40° C. for 60seconds. Subsequently, the PCR reaction solution was heated from 40° C.to 95° C. at a rate of temperature rise of 1° C./3 seconds, and thechange in fluorescence intensity over time was measured. The measurementwavelength was 450 to 480 nm (for detection of the fluorescent dye,Pacific Blue), 515 to 555 nm (for detection of the fluorescent dye,BODIPY FL), and 585 to 700 nm (for detection of the fluorescent dye,TAMRA). The time required for 50 cycles of PCR was approximately onehour.

TABLE 6 <PCR reaction solution; unit: μl> Distilled water 19.875 5% NaN₃0.5 20% BSA 1 40% Glycerol 3.125 10 × Gene Taq buffer * 5 2.5 mM dNTPs 4100 mM MgCl₂ 1 5 μM probe for UGT1A1*6 1 5 μM probe 1 for UGT1A1*27 0.55 μM probe 2 for UGT1A1*27 1 5 μM probe for UGT1A1*28 0.5 100 μMUGT1A1*6 F1 primer 0.25 100 μM UGT1A1*6 R1 primer 0.5 100 μM UGT1A1*27F2 primer 0.25 100 μM UGT1A1*27 R2 primer 0.5 100 μM UGT1A1*28 F3 primer0.25 100 μM UGT1A1*28 R3 primer 0.5 5 U/μl Gene Taq FP * 0.25 Total 40μL * Trade name, Gene Taq Fp: manufactured by Nippon Gene Co., Ltd.<Probes>

Probe for UGT1A1*6 (SEQ ID NO: 55)5′-agagacagagcattttacac-(Pacific Blue)-3′ Probe 1 for UGT1A1*27(SEQ ID NO: 62) 5′-ttattcccagtatgcaaccc-(TAMRA)-3′ Probe 2 for UGT1A1*27(SEQ ID NO: 62) 5′-ttattcccagtatgcaacc-P-3′ Probe for UGT1A1*28(SEQ ID NO: 69) 5′-(BODIPY FL)-ccatatatatatatatataagtaggagag-P-3′<Primer Set>

UGT1A1*6 F1 primer 5′-agcagaggggacatgaaata-3′ (SEQ ID NO: 4)UGT1A1*6 R1 primer 5′-aacattatgcccgagactaac-3′ (SEQ ID NO: 13)UGT1A1*27 F2 primer 5′-agaactttctgtgcgacg-3′ (SEQ ID NO: 21)UGT1A1*27 R2 primer 5′-cagatgcagagctcaatagg-3′ (SEQ ID NO: 29)UGT1A1*28 F3 primer 5′-gtcacgtgacacagtcaaac-3′ (SEQ ID NO: 42)UGT1A1*28 R3 primer 5′-cattgctcctgccagag-3′ (SEQ ID NO: 48)

The Tm value of a hybrid that matches with the probe for UGT1A1*6 is 63°C. and that of a hybrid that mismatches therewith is 56° C., the Tmvalue of a hybrid that matches with the probe for UGT1A1*27 is 61° C.and that of a hybrid that mismatches therewith is 56° C., and the Tmvalue of a hybrid that matches with the probe for UGT1A1*28 is 58° C.and that of a hybrid that mismatches therewith is 54° C.

The results for Samples 1 to 9 are indicated in FIGS. 1 to 3. Thesefigures show graphs of Tm analysis that indicate the changes influorescence intensity accompanying temperature rise. The differentialvalue of the vertical axis indicates “−d fluorescence intensityincrease/dt”, while the horizontal axis indicates temperature (the sameapplies below). As shown in these graphs, the genotypes of UGT1A1*6,UGT1A1*27, and UGT1A1*28 in each sample were determined from the peaksof the signals. In order to support the results of these examples, withrespect to nine subjects, the genotypes of UGT1A1*6, UGT1A1*27, andUGT1A1*28 were confirmed by the RFLP method and the sequencing method.As a result, the same results as those obtained in the example wereobtained. Accordingly, the use of a primer set of the present inventionmade it possible to amplify three regions of the UGT1A1 genesimultaneously in the same reaction solution using a whole blood samplethat had not been pretreated and to analyze the three types ofpolymorphisms using the same reaction solution.

EXAMPLE 2

Blood was collected from two subjects using EDTA blood collection tubes(Samples 1 and 2). Subsequently, 10 μL of blood thus obtained and 70 μLof diluent A described below were mixed together. Further, 10 μL of thismixture and 70 μL of diluent B described below were mixed together.Subsequently, 10 μL of the mixture thus obtained was heat-treated at 95°C. for five minutes. Thereafter, this was added to 46 μL of PCR reactionsolution having the following composition, and then PCR was performedusing a thermal cycler. Conditions for PCR were as follows. That is,after treating at 95° C. for 60 seconds, one cycle of treatment at 95°C. for 1 second and at 60° C. for 15 seconds was repeated for 50 cycles,and further it was treated at 95° C. for 1 second and at 40° C. for 60seconds. Subsequently, the PCR reaction solution was heated from 40° C.to 75° C. at a rate of temperature rise of 1° C./3 seconds, and thechange in fluorescence intensity over time was measured. The measurementwavelength was 450 to 480 nm (for detection of the fluorescent dye,Pacific Blue), 515 to 555 nm (for detection of the fluorescent dye,BODIPY FL), and 585 to 700 nm (for detection of the fluorescent dye,TAMRA).

<Diluent A>

-   10 mM Tris-HCl (pH 8), 0.1 mM EDTA, 0.05% NaN3, 0.3% SDS    <Diluent B>-   10 mM Tris-HCl (pH 8), 0.1 mM EDTA, 0.05% NaN₃

TABLE 7 <PCR reaction solution; unit: μl> Distilled water 15 5% NaN₃ 0.520% BSA 0.5 40% Glycerol 12.5 10 × Gene Taq buffer * 5 2.5 mM dNTPs 4 5μM probe for UGT1A1*6 2 5 μM probe for UGT1A1*27 2 5 μM probe forUGT1A1*28 2 100 μM UGT1A1*6 F1 primer 0.25 100 μM UGT1A1*6 R1 primer 0.5100 μM UGT1A1*27 F2 primer 0.5 100 μM UGT1A1*27 R2 primer 0.25 100 μMUGT1A1*28 F3 primer 0.25 100 μM UGT1A1*28 R3 primer 0.5 5 U/μl Gene TaqFP * 0.25 Total 46 μL * Trade name, Gene Taq Fp: manufactured by NipponGene Co., Ltd.<Probes>

Probe for UGT1A1*6 (SEQ ID NO: 56)5′-gagacagagcattttacac-(Pacific Blue)-3′ Probe for UGT1A1*27(SEQ ID NO: 102) 5′-gttpatacTgggaataaac-(TAMRA)-3′ Probe for UGT1A1*28(SEQ ID NO: 69) 5′-(BODIPY FL)-ccatatatatatatatataagtaggagag-P-3′<Primer Set>

UGT1A1*6 F1 primer (SEQ ID NO: 81) 5′-tgaaatagttgtcctagcacctgacgc-3′UGT1A1*6 R1 primer (SEQ ID NO: 91) 5′-caaaagactatttcacatcctccctttgg-3′UGT1A1*27 F2 primer (SEQ ID NO: 92) 5′-ccttttcacagaactttctgtgcgacg-3′UGT1A1*27 R2 primer (SEQ ID NO: 98) 5′-gccagacagatgcagagctcaatagg-3′UGT1A1*28 F3 primer (SEQ ID NO: 123)5′-agatttttatagtcacgtgacacagtcaaac-3′ UGT1A1*28 R3 primer(SEQ ID NO: 46) 5′-cgcctttgctcctgccagag-3′

The Tm value of a hybrid that matches with the probe for UGT1A1*6 is 57°C. and that of a hybrid that mismatches therewith is 50° C., the Tmvalue of a hybrid that matches with the probe for UGT1A1*27 is 57° C.and that of a hybrid that mismatches therewith is 50° C., and the Tmvalue of a hybrid that matches with the probe for UGT1A1*28 is 53° C.and that of a hybrid that mismatches therewith is 49° C.

Results of Samples 1 and 2 are indicated in FIG. 4. FIG. 4 shows graphsof Tm analysis that indicate the changes in fluorescence intensityaccompanying temperature rise. The differential value of the verticalaxis indicates “−d fluorescence intensity increase/dt”, while thehorizontal axis indicates temperature. As shown in these graphs, thegenotypes of UGT1A1*6, UGT1A1*27, and UGT1A1*28 in each sample weredetermined from the peaks of the signals. In order to support theresults of these examples, with respect to two subjects, the genotypesof UGT1A1*6, UGT1A1*27, and UGT1A1*28 were confirmed by the RFLP methodand the sequencing method. As a result, the same results as thoseobtained in the example were obtained. Accordingly, the use of a primerset of the present invention made it possible to amplify three regionsof the UGT1A1 gene simultaneously in the same reaction solution using awhole blood sample that had not been pretreated and to analyze the threetypes of polymorphisms using the same reaction solution.

INDUSTRIAL APPLICABILITY

As described above, the primer set of the present invention makes itpossible to amplify specifically and efficiently a region including asite where a particular polymorphism (UGT1A1*28. UGT1A1*6, or UGT1A1*27)is generated in the UGT1A1 gene. This allows time and cost to bereduced, which is different from the conventional methods as describedabove. Furthermore, as described above, since the region including asite to be detected of a polymorphism is amplified specifically, forexample, the use of a probe complementary to a sequence to be detectedincluding the site to be detected makes it possible to perform Tmanalysis directly using the aforementioned reaction solution to type thepolymorphism. Moreover, since amplification and typing can be carriedout using one reaction solution, the operation can be automated. The useof the primer set of the present invention allows a pretreatment to beomitted even in the case of, for example, a contaminated sample (forinstance, whole blood or oral mucosa), and therefore the amplificationreaction can be carried out quicker and more easily. Furthermore, whenthe primer set of the present invention is used, the amplificationreaction can be carried out with higher amplification efficiency ascompared to conventional cases and thus the reaction time can also beshortened. According to the primer set of the present invention, thereagent including the same, as well as the method of manufacturing anamplification product using them, since the polymorphism in the UGT1A1gene can be analyzed quickly and simply, it can be said that they areconsiderably effective in the field of medicine.

[Sequence Table] TF07037-01.ST25.txt

The invention claimed is:
 1. An isolated probe comprising a fluorescentlabel and oligonucleotide (3), wherein the oligonucleotide (3) consistsof a base sequence of SEQ ID NO: 69, and the fluorescent label iscovalently bonded to the oligonucleotide (3).
 2. The probe according toclaim 1, wherein the probe can hybridize to a site in a promoter regioncontaining a polymorphism which regulates expression of UGT1A1 (uridinediphosphate glucuronosyl transferase 1 family, polypeptide A1) gene. 3.The probe according to claim 2, wherein the polymorphism indicatessusceptibility to side effects of an anticancer agent in a subject. 4.The probe according to claim 1, wherein the probe has a meltingtemperature Tm between 40 and 50° C.
 5. A reagent compositioncomprising: the probe according to claim 1; and at least one of a probecomprising oligonucleotide (1) and a probe comprising oligonucleotide(2), wherein the oligonucleotide (1) consists of a base sequenceselected from a group consisting of SEQ ID NO: 51 to 58, and theoligonucleotide (2) comprises at least one of oligonucleotides (2-1) and(2-2), wherein the oligonucleotide (2-1) consists of a base sequenceselected from a group consisting of SEQ ID NO: 59 to 66, and theoligonucleotide (2-2) consists of a base sequence selected from a groupconsisting of SEQ ID NO: 100 to
 105. 6. The reagent compositionaccording to claim 5, wherein the reagent comprises the probe accordingto claim 1, the probe composed of the oligonucleotide (1), and the probecomposed of the oligonucleotide (2).
 7. The reagent compositionaccording to claim 5, wherein the oligonucleotide (1) is at least one ofoligonucleotide consisting of the base sequence of SEQ ID NO: 55 andoligonucleotide consisting of the base sequence of SEQ ID NO: 56, theoligonucleotide (2-1) is oligonucleotide consisting of the base sequenceof SEQ ID NO: 62, and the oligonucleotide (2-2) is oligonucleotideconsisting of the base sequence of SEQ ID NO:
 102. 8. The reagentcomposition according to claim 5, further comprising primer set (3) foramplifying the UGT1A1 gene, wherein the primer set (3) is a primer setof a pair of primers including a forward primer comprisingoligonucleotide (F3) and a reverse primer comprising oligonucleotide(R3), wherein the oligonucleotide (F3) consists of a base sequenceselected from a group consisting of SEQ ID NO: 33 to 45 and SEQ ID NO:123 to 124, and the oligonucleotide (R3) consists of a base sequenceselected from a group consisting of SEQ ID NO: 46 to
 50. 9. The reagentcomposition according to claim 8, further comprising at least one ofprimer sets (1) and (2) for amplifying the UGT1A1 gene, wherein theprimer set (1) is a primer set of a pair of primers including a forwardprimer composed of the following oligonucleotide (F1) and a reverseprimer composed of the following oligonucleotide (R1), wherein theoligonucleotide (F1) consists of a base sequence selected from a groupconsisting of SEQ ID NO: 2 to 6 and SEQ ID NO: 74 to 90, and theoligonucleotide (R1) consists of a base sequence selected from a groupconsisting of SEQ ID NO: 7 to 17, SEQ ID NO: 91, and SEQ ID NO: 106 to122, and the primer set (2) is a primer set of a pair of primersincluding a forward primer composed of the following oligonucleotide(F2) and a reverse primer composed of the following oligonucleotide(R2), wherein the oligonucleotide (F2) consists of a base sequenceselected from a group consisting of SEQ ID NO: 18 to 24 and SEQ ID NO:92 to 97, and the oligonucleotide (R2) consists of a base sequenceselected from a group consisting of SEQ ID NO: 25 to 32 and SEQ ID NO:98 to
 99. 10. The reagent composition according to claim 5, wherein theprobe comprising the oligonucleotide (1) can hybridize to a nucleotidesequence encoding a UGT1A1 gene containing a polymorphism at base 2160of SEQ ID NO: 1, and the probe comprising the oligonucleotide (2) canhybridize to a nucleotide sequence encoding a UGT1A1 gene containing apolymorphism at base 2635 of SEQ ID NO:
 1. 11. The reagent compositionaccording to claim 10, wherein the polymorphism indicates susceptibilityto side effects of an anticancer agent in a subject.
 12. The reagentcomposition according to claim 5, wherein the probe comprising theoligonucleotide (1) has a melting temperature Tm between 51-58° C., andthe probe comprising the oligonucleotide (2) has a melting temperatureTm between 59-105° C.
 13. A polymorphism analysis method of analyzing apolymorphism of a site to be detected in the UGT1A1 gene, wherein themethod comprises the following processes (i) to (iv): (i) amplifying aregion including a site to be detected in the UGT1A1 gene in a reactionsolution with nucleic acid contained in a sample being used as atemplate, (ii) preparing a reaction solution that contains theamplification product obtained in the process (i) and the probeaccording to claim 1, (iii) measuring signal values that indicate moltenstates of a hybridization product between the amplification product andthe probe while changing the temperature of the reaction solution, and(iv) determining a polymorphism of the site to be detected from a changein the signal values accompanying a change in the temperature.
 14. Thepolymorphism analysis method according to claim 13, wherein, in theprocess (i), the probe is added to the reaction solution prior to anamplification reaction.
 15. The polymorphism analysis method accordingto claim 13, wherein the sample is a biological sample.
 16. Thepolymorphism analysis method according to claim 15, wherein thebiological sample is whole blood.
 17. The polymorphism analysis methodaccording to claim 13, wherein, in the process (ii), the reactionsolution further contains at least one of a probe composed of thefollowing oligonucleotide (1) and a probe composed of the followingoligonucleotide (2), wherein the oligonucleotide (1) consists of a basesequence selected from a group consisting of SEQ ID NO: 51 to 58, theoligonucleotide (2) comprises at least one of oligonucleotides (2-1) and(2-2), wherein the oligonucleotide (2-1) consists of a base sequenceselected from a group consisting of SEQ ID NO: 59 to 66, and theoligonucleotide (2-2) consists of a base sequence selected from a groupconsisting of SEQ ID NO: 100 to
 105. 18. The polymorphism analysismethod according to claim 17, wherein the reaction solution contains theprobe composed of the oligonucleotide (1), and the probe composed of theoligonucleotide (2).
 19. The polymorphism analysis method according toclaim 17, wherein the oligonucleotide (1) is at least one ofoligonucleotide consisting of the base sequence of SEQ ID NO: 55 andoligonucleotide consisting of the base sequence of SEQ ID NO: 56, theoligonucleotide (2-1) is oligonucleotide consisting of the base sequenceof SEQ ID NO: 62, and the oligonucleotide (2-2) is oligonucleotideconsisting of the base sequence of SEQ ID NO:
 102. 20. The polymorphismanalysis method according to claim 13, wherein, in the process (i), theamplification of the UGT1A1 gene is carried out in the reaction solutionusing primer set (3), wherein the primer set (3): is a primer set of apair of primers including a forward primer comprising oligonucleotide(F3) and a reverse primer comprising oligonucleotide (R3), wherein theoligonucleotide (F3) consists of a base sequence selected from a groupconsisting of SEQ ID NO: 33 to 45 and SEQ ID NO: 123 to 124, and theoligonucleotide (R3) consists of a base sequence selected from a groupconsisting of SEQ ID NO: 46 to
 50. 21. The polymorphism analysis methodaccording to claim 20, wherein the primer set (3) is a primer set of apair of primers including a forward primer comprising oligonucleotide(F3′) and a reverse primer comprising oligonucleotide (R3′), wherein theoligonucleotide (F3′) comprises at least one of oligonucleotideconsisting of the base sequence of SEQ ID NO: 42 and oligonucleotideconsisting of the base sequence of SEQ ID NO: 123, and theoligonucleotide (R3′) comprises at least one of oligonucleotideconsisting of the base sequence of SEQ ID NO: 46 and oligonucleotideconsisting of the base sequence of SEQ ID NO:
 48. 22. The polymorphismanalysis method according to claim 20, wherein, in the process (i), theamplification of the UGT1A1 gene is carried out in the same reactionsolution using the primer set (3) and at least one of primer set (1) andprimer set (2), wherein the primer set (1) is a primer set of a pair ofprimers including a forward primer composed of the followingoligonucleotide (F1) and a reverse primer composed of the followingoligonucleotide (R1), wherein the oligonucleotide (F1) consists of abase sequence selected from a group consisting of SEQ ID NO: 2 to 6 andSEQ ID NO: 74 to 90, and the oligonucleotide (R1) consists of a basesequence selected from a group consisting of SEQ ID NO: 7 to 17, SEQ IDNO: 91, and SEQ ID NO: 106 to 122, and the primer set (2) is a primerset of a pair of primers including a forward primer composed of thefollowing oligonucleotide (F2) and a reverse primer composed of thefollowing oligonucleotide (R2):, wherein the oligonucleotide (F2)consists of a base sequence selected from a group consisting of SEQ IDNO: 18 to 24 and SEQ ID NO: 92 to 97, and the oligonucleotide (R2)consists of a base sequence selected from a group consisting of SEQ IDNO: 25 to 32 and SEQ ID NO: 98 to
 99. 23. The polymorphism analysismethod according to claim 22, wherein, in the process (i), theamplification of the UGT1A1 gene is carried out in the same reactionsolution using the primer set (3), the primer set (1), and the primerset (2).
 24. The polymorphism analysis method according to claim 23,wherein the primer set (1) is primer set (1′) and the primer set (2) isprimer set (2′), wherein the primer set (1′) is a primer set of a pairof primers including a forward primer comprising oligonucleotide (F1′)and a reverse primer comprising oligonucleotide (R1′), wherein theoligonucleotide (F1′) comprises at least one of oligonucleotideconsisting of the base sequence of SEQ ID NO: 4 and oligonucleotideconsisting of the base sequence of SEQ ID NO: 81, and theoligonucleotide (F1′) comprises at least one of oligonucleotideconsisting of the base sequence of SEQ ID NO: 13 and oligonucleotideconsisting of the base sequence of SEQ ID NO: 91, and the primer set(2′) is a primer set of a pair of primers including a forward primercomprising oligonucleotide (F2′) and a reverse primer comprisingoligonucleotide (R2′), wherein the oligonucleotide (F2′) comprises atleast one of oligonucleotide consisting of the base sequence of SEQ IDNO: 21 and oligonucleotide consisting of the base sequence of SEQ ID NO:92, and the oligonucleotide (R2′) comprises at least one ofoligonucleotide consisting of the base sequence of SEQ ID NO: 29 andoligonucleotide consisting of the base sequence of SEQ ID NO: 98.